Adult stem cells possess an immense, intrinsic capacity for proliferation and yet avoid the immortality associated with neoplastic growth. The genetic pathways that control this "self-renewal" in stem cells are unknown, but key, "master regulators" of such programs have been described for hematopoietic and spermatogonial stem cells. A corresponding master regulator of stratified epithelia, including epidermis, breast, and prostate, had remained elusive, exposing a major gap in our understanding of these highly regenerative and cancer-prone tissues. However, recent data identify this missing epithelial stem cell regulator as p63, a member of the p53 family of transcription factors. Disconcertingly, whole-genome ChIp-on-chip data indicate that p63 directly regulates more than 800 genes, a hint that genetic programs underlying self-renewal in stem cells are likely complex and resistant to analysis. In this proposal, we develop the hypothesis that self- renewal is controlled by common mechanisms in stem cells of different lineages, despite the looming contradiction that the known master regulators of self-renewal are tissue-specific. Key to our hypothesis is that these master regulators must coordinate three genetic programs to maintain stem cells, two of which dictate their tissue specificity. Thus in addition to controlling a self-renewal program, these master regulators also need to drive interactions with the stem cell niche as well as forestall tissue-specific differentiation programs. To identify the putative common genetic program underlying self-renewal, we establish experimental strategies in four aims that take advantage of the unique clonogenic properties of epithelial stem cells. In Aim 1, we identify genes involved in self-renewal by "masking" the tissue-specific roles of p63. To do this, we compare the gene expression profiles of phenotypically similar, epithelial stem cells of the same lineage that differ only in their proliferation history. In Aim 2, we identify the genes directly regulated by Bmi-1 in hematopoietic cells and compare these with those regulated by p63 in epithelial stem cells to search for mutually regulated subsets of genes that comprise a common program of self-renewal. In Aim 3, we approach this same question by screening shRNAs against a subset of transcription factors regulated by p63 for their representation over replicative time. Our goal here is to use all three datasets from Aims 1, 2, and 3 to generate a restricted list of candidate genes for individual, in-depth analysis of function in self-renewal pathways. In Aim 4, we examine the molecular mechanisms that control p63 stability as this feature ultimately controls self-renewal. We anticipate that these strategies will provide fundamental insights into the putative "common" pathways by which p63 and other, tissue-specific master regulators impart stemness to regenerative cells. PUBLIC HEALTH RELEVANCE: Answers to the questions posed here will unveil information critical to the therapeutic use of adult stem cells in general and of epithelial stem cells in particular. Given that epithelial tissues contribute to the majority of human cancers, this work should also help to unravel how p63-dependent regenerative properties contribute to the process of tumorigenesis.